Fluorescence In Situ Hybridization (FISH) Detailed Notes

#### Fluorescence in Situ Hybridization (FISH) - A procedure used to locate specific DNA sequences on chromosomes, aiding in diagnostics and research. FISH is a molecular cytogenetic technique that allows scientists to visualize and map genetic material in individual cells, providing valuable insights into genome organization and gene expression. - Employs fluorescent probes that bind to specific DNA sequences, allowing for their detection and localization within the chromosome. The probes are labeled with fluorescent dyes, enabling their visualization under a fluorescence microscope and precise localization of target sequences. - Widely used in diagnosing chromosomal abnormalities, such as aneuploidy, translocations, and deletions, which are crucial in genetic counseling and cancer diagnostics. FISH enables the detection of genetic aberrations that are associated with various diseases, facilitating accurate diagnosis and risk assessment. - Depends on the stability of the DNA double helix, as the hybridization process requires intact DNA structure for probe binding. Factors such as temperature, pH, and salt concentration can affect the stability of the DNA double helix and the efficiency of hybridization. ##### In Situ Hybridization - Used to localize DNA sequences on chromosomes, providing insights into gene mapping and chromosomal organization. In situ hybridization techniques enable the study of gene expression patterns and chromosomal architecture in a variety of biological contexts. - Hydrogen bonds holding the helix together can be broken by heat or chemicals, such as formamide, allowing for the separation of DNA strands. Denaturation is a critical step in the hybridization process, as it enables the probe to access and bind to the target DNA sequence. - The helix can re-form under favorable conditions, which is the basis for molecular hybridization, a process where complementary strands anneal. Annealing is driven by the affinity between complementary base pairs and is optimized by controlling temperature, salt concentration, and probe concentration. - Molecular hybridization: A labeled DNA or RNA sequence is used as a probe to identify or quantify its counterpart in a biological sample, enabling gene expression analysis and pathogen detection. Molecular hybridization is a powerful tool for studying gene function and identifying infectious agents in clinical samples. ##### Fluorescent Probes - Fluorescent labels have replaced radioactive labels due to their safety, stability, and ease of detection, making them ideal for clinical and research applications. Fluorescent probes offer several advantages over traditional radioactive probes, including higher resolution, multiplexing capabilities, and reduced health hazards. - A fluorescent DNA sequence acts as a 'magnet' to find a specific DNA sequence, allowing for targeted localization of genetic elements within the genome. The specificity of the probe-target interaction is determined by the sequence complementarity between the probe and the target DNA. - Hybridization requires a probe (fluorescent copy of the DNA sequence of interest) and a target (the DNA sequence to be identified). The specificity of the hybridization depends on the sequence complementarity between the probe and the target. 1. Make a fluorescence/modified copy of the probe sequence using techniques like PCR or enzymatic labeling with fluorescent nucleotides. PCR amplification is a common method for generating large quantities of probe DNA, while enzymatic labeling allows for the incorporation of fluorescent nucleotides into the probe. 2. Denature both the target and probe sequences with heat or chemicals to create single-stranded DNA, facilitating probe binding. Denaturation disrupts the hydrogen bonds between complementary base pairs, allowing the probe to access and hybridize to the target DNA. 3. Mix the probe and target together, allowing the probe to hybridize to its complementary sequence through base pairing. Hybridization is typically performed under controlled conditions to ensure optimal probe binding and minimize non-specific interactions. 4. Detect hybrids using a fluorescent microscope equipped with appropriate filters to visualize the fluorescent signal, and image analysis software to quantify the signal. Fluorescence microscopy allows for the visualization and quantification of fluorescent signals, enabling the detection of specific DNA sequences within the sample. ##### Designing FISH Experiments - Consider the sensitivity and resolution needed for the experiment within the technical limits of fluorescence microscopy to optimize probe design and imaging parameters. Careful planning of FISH experiments is essential for obtaining accurate and reliable results. - Sensitivity: Light-gathering ability of the microscope to detect small target sequences, which can be enhanced by using high-quality optics and sensitive detectors. Sensitivity is affected by factors such as probe labeling efficiency, hybridization conditions, and detector performance. - Resolution: Ability to distinguish between two points along a chromosome, affecting the accuracy of gene mapping and breakpoint detection. Resolution is determined by factors such as numerical aperture of the objective lens, wavelength of light, and sample preparation techniques. - Light microscopy can't resolve objects separated by less than 200-250 nm, limiting the ability to detect closely spaced DNA sequences. This limitation can be overcome by using super-resolution microscopy techniques, which can achieve resolutions beyond the diffraction limit of light. - Chromosome painting can quickly identify chromosomes involved in translocations and large deletions/duplications, but not small ones, making it suitable for karyotyping and screening. Chromosome painting is a valuable tool for detecting chromosomal abnormalities in a variety of clinical and research settings. ##### Different FISH Techniques - Diversification of FISH is due to improvements in sensitivity, specificity, and resolution, expanding its applications in various fields. The development of new FISH techniques has enabled the study of various biological processes at the molecular level. - Centromere-FISH (ACM-FISH): Detects chromosomal abnormalities in sperm cells; used to explain infertility in oligozoospermic men, providing insights into male reproductive health. ACM-FISH is a valuable tool for assessing sperm quality and identifying potential causes of male infertility. - armFISH: Detects chromosomal abnormalities in the p- and q-arms of all 24 human chromosomes, except the p-arm of the Y and acrocentric chromosomes, aiding in comprehensive chromosomal analysis. armFISH provides a comprehensive assessment of chromosomal integrity and can detect subtle chromosomal aberrations. - Consider the conformation of DNA within the chromosome. The degree of compaction affects probe accessibility and hybridization efficiency. - Metaphase chromosomes are highly compacted, requiring harsher denaturation conditions and longer hybridization times. Metaphase FISH is typically used for high-resolution mapping of DNA sequences on chromosomes. - Interphase chromosomes are less compacted than metaphase chromosomes but more compacted than naked DNA, allowing for faster hybridization kinetics. Interphase FISH is useful for studying gene expression and chromosomal organization in non-dividing cells. ##### Using FISH to Identify Gene Positions - FISH is a powerful tool for locating cloned DNA sequences on metaphase chromosomes, enabling the creation of high-resolution genetic maps. FISH is used to physically map genes on chromosomes, providing valuable information about genome organization and gene function. - FISH results are crucial in mapping genes on human chromosomes, contributing to our understanding of genome organization and gene function. The Human Genome Project relied heavily on FISH mapping data to construct physical maps of the human genome. - Results are compiled into databases for the Human Genome Project (HGP), providing valuable resources for researchers and clinicians. These databases provide access to a wealth of information about gene location, structure, and function. - Current FISH applications are mainly for clinical diagnoses, such as prenatal genetic screening, cancer cytogenetics, and infectious disease detection. FISH is widely used in clinical laboratories for the diagnosis and management of a variety of diseases. ##### Diagnosing Chromosomal Abnormalities - FISH is important in diagnosing chromosomal abnormalities like deletions, duplications, and translocations, aiding in genetic counseling and personalized medicine. FISH is used to identify chromosomal abnormalities that are associated with genetic disorders and cancer, facilitating accurate diagnosis and personalized treatment strategies. ##### Using FISH Probes to Paint Chromosomes - Multicolor FISH (spectral karyotyping) is used to scan metaphase chromosomes for rearrangements, enabling the detection of complex chromosomal abnormalities in cancer cells. Multicolor FISH allows for the simultaneous visualization of multiple chromosomes in different colors, facilitating the detection of chromosomal rearrangements and aneuploidies. - Multifluor FISH generates a karyotype where each chromosome has a different color, facilitating the identification of chromosomal rearrangements and aneuploidies. This technique is valuable for analyzing complex chromosomal aberrations in cancer cells and other genetically abnormal cells. - Each "paint" is a collection of hybridization probes for sequences along a specific chromosome, allowing for comprehensive chromosomal coverage. Chromosome paints are used to visualize entire chromosomes, facilitating the detection of large-scale chromosomal abnormalities. - Digital processing distinguishes spectral differences between chromosomes, enhancing the resolution and accuracy of karyotyping. Digital image analysis techniques are used to enhance the visualization and quantification of fluorescent signals in multicolor FISH experiments. - Normal chromosomes have a uniform color, while rearranged chromosomes have a striped appearance, indicating structural abnormalities. The presence of striped chromosomes in a multicolor FISH karyotype indicates the presence of chromosomal rearrangements, such as translocations and inversions. ##### Other FISH Techniques - Multilocus or ML-FISH. - Catalyzed Reporter Deposition-FISH (CARD-FISH): Useful for detecting, identifying, and quantifying microorganisms in bioleaching processes, providing insights into microbial ecology and environmental biotechnology. CARD-FISH is a sensitive technique for detecting and quantifying specific microbial populations in environmental samples. - Cellular Compartment Analysis of Temporal (Cat) and Activity by FISH (catFISH): Used to determine interactions of neuronal populations associated with different behaviors, aiding in neuroscience research. catFISH is a powerful tool for studying neuronal activity and connectivity in the brain. - Cytochalasin B (CB-FISH): Involves hybridization oon binucleated cells in which cytokinesis has been blocked by treatment with cytochalasin B, enabling the study of chromosome segregation errors. CB-FISH is used to study chromosome segregation errors and the mechanisms of aneuploidy. - Chromosome Orientation (CO)-FISH: Determines the relative orientation of two or more DNA sequences along a chromosome; useful in assessing chromosomal translocations and inversions, allowing for precise mapping of genomic rearrangements. CO-FISH provides information about the orientation of DNA sequences along a chromosome, which is useful for mapping genomic rearrangements. - Combined Binary Ration (COBRA)-FISH: Enables recognition of all human chromosome arms based on color and mapping of gene and viral integration sites in the context of chromosome arm painting, facilitating comprehensive genomic analysis. COBRA-FISH is used for comprehensive genomic analysis and mapping of gene and viral integration sites. - Reverse-FISH: Useful for characterizing marker chromosomes and chromosome amplifications in cancer, aiding in cancer diagnostics and prognostics. Reverse-FISH is used to identify the origin of marker chromosomes and to characterize chromosome amplifications in cancer cells. ##### FISH Basics - Uses artificial DNA segments with fluorescent molecules attached (probes) to target and visualize specific DNA sequences. FISH relies on the ability to design and synthesize DNA probes that are complementary to specific DNA sequences of interest. - Probes target specific areas of chromosomes, enabling the detection of chromosomal abnormalities and gene mapping. The specificity of the probe-target interaction is critical for accurate detection of chromosomal abnormalities and gene mapping. - Hybridization: Probe binds to complementary DNA from the patient's sample, forming a stable duplex that can be detected using fluorescence microscopy. The hybridization process is influenced by factors such as temperature, salt concentration, and probe concentration. - Binding causes the probe to glow, signaling chromosome abnormalities such as deletions, duplications, and translocations. The intensity of the fluorescent signal is proportional to the amount of target DNA present in the sample. - If there are extra copies of a gene, there will be more glowing signals, indicating gene amplification. Gene amplification can be associated with increased expression of the amplified gene, contributing to disease pathogenesis. ##### Types of Abnormalities Detected - Duplication/amplification: Extra copies of chromosomes, chromosome parts, or genes, which can contribute to genetic disorders and cancer. Duplications and amplifications can lead to increased expression of genes located in the amplified region, contributing to disease development. - Deletion: Missing chromosomes or chromosome parts, leading to loss of genetic material and potential developmental defects. Deletions can result in the loss of essential genes, leading to developmental defects and other health problems. - Translocation: Part of one chromosome detaches and reattaches to another chromosome, disrupting gene expression and causing genetic imbalances. - Example: Philadelphia chromosome rearrangement in acute lymphoblastic leukemia (ALL), where part of chromosome 9 attaches to chromosome 22, and vice versa, leading to the formation of the BCR-ABL fusion gene. The BCR-ABL fusion gene encodes a constitutively active tyrosine kinase that drives uncontrolled cell proliferation in ALL. ##### ML-FISH - Simultaneous use of multiple probes in multicolor FISH to detect multiple targets simultaneously. ML-FISH allows for the simultaneous detection of multiple genetic targets, providing a comprehensive assessment of genomic alterations. - Initially designed to screen for multiple microdeletion syndromes in patients with unexplained developmental delay and/or mental retardation, providing a comprehensive genetic screening approach. ML-FISH is a valuable tool for identifying microdeletion syndromes in children with developmental delay and mental retardation. ##### Dual-Color FISH - Used to detect gene amplification of EGFR and CDK4 in brain cancer tumors, aiding in the identification of potential therapeutic targets. Dual-color FISH allows for the simultaneous detection of two different genetic targets, providing valuable information about gene amplification and chromosomal abnormalities. - Uses two fluorescent probes: one for the EGFR gene and another for the CDK4 gene or a chromosome 7 centromere control, allowing for normalization and accurate quantification of gene copy number. The use of a control probe allows for normalization of the FISH signal, ensuring accurate quantification of gene copy number. - The ratio of signals from the gene-specific probes to the control probe determines if amplification has occurred, which is used to guide treatment decisions. FISH results can be used to guide treatment decisions in cancer patients, ensuring that they receive the most appropriate therapy. ##### Probes - Artificial DNA strand with fluorescence that hybridizes with real DNA, allowing for specific targeting of genomic regions. Probes are designed to be complementary to specific DNA sequences, allowing for targeted detection of genomic regions. - Attaches to complementary DNA strand and lights up where there are abnormalities, providing visual confirmation of genetic alterations. The fluorescent signal emitted by the probe indicates the presence of the target DNA sequence and can be used to detect genetic alterations. - FISH: A probe is an artificial DNA strand with fluorescence that is used as a complementary strand to an original DNA strand/the strand you are testing, enabling the detection of specific DNA sequences. - Probes bind to specific DNA sequences on chromosomes and are labeled with fluorescent dyes for visualization under a fluorescence microscope, facilitating the identification of genetic abnormalities. The choice of fluorescent dye depends on the specific application and the available instrumentation. - Hybridizes with the original DNA strand and matches up base pairs, forming a stable double helix. The stability of the hybrid is influenced by factors such as temperature, salt concentration, and probe sequence. - Where there are not matches or abnormalities, the fluorescent strand (probe) will glow, indicating deletions, amplifications, or translocations, providing valuable diagnostic information. The pattern of fluorescent signals provides information about the presence and nature of genetic abnormalities. - Provides information on gene location, copy number, and abnormalities, which is crucial for understanding disease mechanisms and developing targeted therapies. FISH is a versatile tool that can be used to study a wide range of biological processes. - OMIM- database website for information on genes and genetic disorders. ##### Research Areas - EGFR: Studying its role in cancer development and as a therapeutic target. - Chromothripsis & how it forms: Understanding the mechanisms and consequences of chromosomal shattering and rearrangement. - CDK4: Investigating its involvement in cell cycle regulation and as a target for cancer therapy. - Micronuclei: Exploring their role in genomic instability and cancer progression. ##### EGFR - A protein that helps cells grow and divide, playing a crucial role in cell signaling pathways. $EGFR$ is a transmembrane receptor that binds to epidermal growth factor (EGF) and other growth factors, initiating downstream signaling cascades. - A receptor protein on the surface of cells that receives signals from epidermal growth factor (EGF), triggering downstream signaling cascades. Upon ligand binding, $EGFR$ undergoes dimerization and autophosphorylation, activating intracellular signaling pathways. - In glioblastoma, $EGFR$ mutations or amplification can lead to uncontrolled cell growth and development of tumors, contributing to the aggressive nature of the disease. $EGFR$ is one of the most frequently amplified genes in glioblastoma, driving tumor growth and resistance to therapy. - Mutations to growth factor receptors can lead to their constant activation, which can cause uncontrolled cell division – a hallmark of cancer– as the cell keeps receiving signals to grow, disrupting normal cellular processes. Constitutive activation of growth factor receptors can lead to aberrant signaling, promoting cell proliferation, survival, and migration. - In glioblastoma, specifically, a growth factor receptor named $EGFR$ is commonly mutated or amplified. In fact, it is altered in approximately 60% of GBM tumors, making it a key therapeutic target. - EGRFIII mutation could change the way a GMA cells use their signaling networks to evade treatment and stay activated. This mutation also could cause the reprogramming of tumor cells’ metabolism, which allows the tumor to take in the energy it needs to grow uncontrollably, affecting treatment response. - Clinical trials trying to target $EGFR$ have failed as an effective treatment, highlighting the complexity of $EGFR$ signaling and the need for alternative therapeutic approaches. Resistance to $EGFR$ inhibitors can arise through various mechanisms, including bypass signaling and downstream mutations. ##### Chromothripsis - Catastrophic event of chromosomal shattering and reassembly, leading to $EGFR$ amplification and the formation of ecDNA, which promotes tumorigenesis. Chromothripsis is often associated with genomic instability and can contribute to the rapid evolution of cancer genomes. - $EGFR$ amplification via ecDNA is likely initiated as a single event where genomic regions overlapping the $EGFR$ gene are joined via non-homologous end joining and are subsequently clonally amplified, resulting in high levels of $EGFR$ expression. ecDNA allows for rapid and flexible amplification of oncogenes, promoting tumor growth and adaptation to changing environments. - A phenomenon where chromosomes are shattered into fragments, and then the fragments are haphazardly rejoined, resulting in a high number of complex rearrangements within a single cell, contributing to genomic instability and cancer development. - Alongside chromothripsis, an event has been identified, called chromoanasynthesis. The chromoanasynthesis based on a disorder of the replication process—a sequential stop of replication forks or a violation of replication mechanisms mediated by microhomology an error-prone repair mechanism that involves alignment of microhomologous sequences internal to the broken ends before joining, and is associated with deletions and insertions that mark the original break sight). - Chomothripsis can cause the $EGFR$ gene to be amplified, meaning there are multiple copies of the gene. The fragmented DNA resulting from chromothripsis can be reassembled into circular DNA structures called ecDNA. A specific type of $EGFR$ variant, $EGFRvIII$ ##### CDK4 - A cyclin-dependent kinase that plays a crucial role in cell cycle regulation and is often deregulated in cancers, including brain cancer, contributing to uncontrolled cell proliferation. $CDK4$ forms a complex with cyclin D, which phosphorylates the retinoblastoma protein (Rb) and promotes cell cycle progression. - Helps drive cells from G1 phase to the S phase of the cell cycle, which is necessary for DNA synthesis and cell division, ensuring proper cell cycle progression. Disruption of $CDK4$ activity can lead to cell cycle arrest and inhibition of cell proliferation. - In brain cancers, $CDK4$ is frequently overexpressed or altered, contributing to uncontrolled cell growth and tumor development, making it a potential therapeutic target. Overexpression of $CDK4$ can drive uncontrolled cell proliferation and contribute to tumor development. - Due to its involvement in cell cycle regulation and its frequent deregulation in brain cancer, $CDK4$ has become a target for therapeutic interventions, such as $CDK4/6$ inhibitors. These inhibitors work by blocking $CDK4$ and $CDK6$, disrupting the cell cycle - and slowing down tumor growth. Some $CDK4/6$ inhibitors have shown promise in treating brain metastases and even certain subtypes of primary brain tumors - The kinase activity of $CDK$-cyclin complexes is also highly controlled by an abundance of CKIs, which serve as brakes to control cell-cycle progression according to the conditions in cells ##### Micronuclei - Cytoplasmic structures containing entire chromosomes or chromosomal fragments, and they result from errors in segregation during mitosis, contributing to genomic instability. Micronuclei are formed when chromosomes or chromosome fragments fail to be incorporated into the main nucleus during cell division. - Small, extra-nuclear structures containing fragmented or mis-segregated chromosomes, reflecting mitotic errors and DNA damage. Micronuclei can arise from a variety of sources, including DNA damage, mitotic errors, and telomere dysfunction. - Contribute to brain cancer development by promoting genomic instability, activating immune responses, and potentially triggering chromothripsis, accelerating tumor progression. Micronuclei can promote genomic instability by releasing DNA fragments into the cytoplasm, which can be incorporated into the main nucleus. - Micronuclei are prone to rupture, releasing DNA fragments into the cytoplasm. This can lead to DNA damage, chromosome rearrangements, and other genomic alterations, which can be hallmarks of cancer. - Micronuclei are often associated with chromothripsis, a catastrophic event that causes extensive genomic rearrangements. This process can lead to the activation of oncogenes and silencing of tumor suppressor genes, promoting tumorigenesis ##### EGFR viii - Mutated version of the epidermal growth factor receptor (EGFR) found in glioblastoma, contributing to tumor aggressiveness and treatment resistance. $vIII$ is a tumor-specific mutation that results in a constitutively active receptor, promoting uncontrolled cell growth. - This mutations is tumor-specific and contributes to cancer development by promoting unregulated cell growth, survival, and invasion, driving tumor progression. - Mutant form of the $EGFR$ receptor that arises for a tumor-specific mutation, specifically an in-frame deletion of exons 2-7. This deletion removes a portion of the receptor’s extracellular domain
\*(removes 801 base pairs & 267 amino acids from the receptor’s extracellular domain), leading to a receptor that cannot bind ligands but is constitutively active, meaning it’s always signaling without the need for an external signal - Egfr viii does not required ligand binding for activation unlike the wild type that needs EGF to bind and trigger - $EGFR$ viii does not require ligand binding for activation due to the deletion of exons 2-7, causing it to autophosphorylate (acts as both the enzyme and the substrate)